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The purpose of this study is to explore the anti-colorectal cancer of Xiaotansanjiefang, a famous traditional Chinese medicine, and its potential anti-cancer mechanism. In this study, the HCT116 cell spheres were prepared as in vitro study model. We found the Xiaotansanjiefang medication was able to inhibit the proliferation of HCT116 cell spheres in a dose-dependent manner, especially in 3 and 6 mg/ml Xiaotansanjiefang medication treated groups. We also found the high concentration of Xiaotansanjiefang medication could suppress the migration and promote the apoptosis of HCT116 cell spheres. Moreover, we found the expression of Jagged 1, Notch 3, Snail, and Hes 1 were decreased in HCT116 cell spheres treated with Xiaotansanjiefang medication. Furthermore, the proliferation and apoptosis behaviors of HCT116 cell spheres treated with Xiaotansanjiefang medication were reversed with the addition of Jagged 1 Fc chimera protein. The expression of Jagged 1, Notch 3, Snail, and Hes 1 were also increased again in HCT116 cells treated with Xiaotansanjiefang medication plus with Jagged 1 Fc chimera protein. The presented study may provide a promising strategy to treat and prevent colorectal cancer.

Mucosecretory lung disease compromises airway epithelial function and is characterized by goblet cell hyperplasia and ciliated cell hypoplasia. Goblet and ciliated cell types are derived from tracheobronchial stem/progenitor cells via a Notch-dependent mechanism. Although specific arrays of Notch receptors regulate cell fate determination, the function of the ligands Jagged1 (JAG1) and JAG2 is unclear. This study examined JAG1 and JAG2 function using human air-liquid-interface cultures that were treated with γ-secretase complex (GSC) inhibitors, neutralizing peptides/antibodies, or WNT/β-catenin pathway antagonists/agonists. These experiments revealed that JAG1 and JAG2 regulated cell fate determination in the tracheobronchial epithelium; however, their roles did not adhere to simple necessity and sufficiency rules. Biochemical studies indicated that JAG1 and JAG2 underwent posttranslational modifications that resulted in generation of a JAG1 C-terminal peptide and regulated the abundance of full-length JAG2 on the cell surface. GSC and glycogen synthase kinase 3 were implicated in these posttranslational events, but WNT agonist/antagonist studies and RNA-Seq indicated a WNT-independent mechanism. Collectively, these data suggest that posttranslational modifications create distinct assemblies of JAG1 and JAG2, which regulate Notch signal strength and determine the fate of tracheobronchial stem/progenitor cells.

Notch signaling is an evolutionarily conserved pathway that functions via direct cell-cell contact. The Notch ligand Jagged1 (Jag1) has been extensively studied in vascular development, particularly for its role in smooth muscle cell maturation. Endothelial cell-expressed Jag1 is essential for blood vessel formation by signaling to nascent vascular smooth muscle cells and promoting their differentiation. Given the established importance of Jag1 in endothelial cell/smooth muscle crosstalk during development, we sought to determine the extent of this communication in the adult vasculature for blood vessel function and homeostasis.We conditionally deleted Jag1 in endothelial cells of adult mice and examined the phenotypic consequences on smooth muscle cells of the vasculature.Our results show that genetic loss of Jag1 in endothelial cells has a significant impact on Notch signaling and vascular smooth muscle function in mature blood vessels. Endothelial cell-specific deletion of Jag1 causes a concomitant loss of JAG1 and NOTCH3 expression in vascular smooth muscle cells, resulting in a transition to a less differentiated state. Aortic vascular smooth muscle cells isolated from the endothelial cell-specific Jag1 deficient mice retain an altered phenotype in culture with fixed changes in gene expression and reduced Notch signaling. Utilizing comparative RNA-sequence analysis, we found that Jag1 deficiency preferentially affects extracellular matrix and adhesion protein gene expression. Vasoreactivity studies revealed a reduced contractile response and impaired agonist-induced relaxation in endothelial cell Jag1-deficient aortas compared to controls.These data are the first to demonstrate that Jag1 in adult endothelial cells is required for the regulation and homeostasis of smooth muscle cell function in arterial vessels partially through the autoregulation of Notch signaling and cell matrix/adhesion components in smooth muscle cells.

Evolutionarily conserved RNA-binding protein Musashi1 (Msi1) can regulate developmentally relevant genes. Here we report the generation and characterization of a mouse model that allows inducible Msi1 overexpression in a temporal and tissue-specific manner. We show that ubiquitous Msi1 induction in ∼5-wk-old mice delays overall growth, alters organ-to-body proportions, and causes premature death. Msi1-overexpressing mice had shortened intestines, diminished intestinal epithelial cell (IEC) proliferation, and decreased growth of small intestine villi and colon crypts. Although Lgr5-positive intestinal stem cell numbers remained constant in Msi1-overexpressing tissue, an observed reduction in Cdc20 expression provided a potential mechanism underlying the intestinal growth defects. We further demonstrated that Msi1 overexpression affects IEC differentiation in a region-specific manner, with ileum tissue being influenced the most. Ilea of mutant mice displayed increased expression of enterocyte markers, but reduced expression of the goblet cell marker Mucin2 and fewer Paneth cells. A higher hairy and enhancer of split 1:mouse atonal homolog 1 ratio in ilea from Msi1-overexpressing mice implicated Notch signaling in inducing enterocyte differentiation. Together, this work implicates Msi1 in mouse postnatal development of multiple organs, with Notch signaling alterations contributing to intestinal defects. This new mouse model will be a useful tool to further elucidate the role of Msi1 in other tissue settings.

Background Notch pathway is highly conserved across species and is involved in the regulation of cell differentiation and activity both in embryonic development and adult life. Notch signaling has an important role in the development of hematopoietic stem cells and their differentiation to committed lineages, as well as in the regulation of several non-hematopoietic cell lines. Objective As Notch signaling has been implicated in various inflammatory and autoimmune diseases, it is of interest to elucidate what role do Notch receptors and ligands have in inflammatory arthritides. Methods We performed a search on the role of Notch receptors (1-4) and Notch ligands Delta-like (DLL) 1, 3, 4 and Jagged (Jag) 1 and 2 in animal models of inflammatory arthritis and most common types of human inflammatory arthritis (rheumatoid arthritis, psoriatic arthritis or ankylosing spondylitis). The initial search identified 135 unique articles, of which 24 were ultimately deemed relevant and included in this systematic review. Results Overall, identified articles describe roles for Notch ligands and receptors in inflammatory arthritis, with Notch activation resulting in enhanced Th1/17 polarization, osteoclast differentiation, macrophage activation and fibroblast-like synoviocyte proliferation. However, the inhibitory role of Notch signaling, especially by Jag1 is also described. Conclusion There is evidence that Notch pathway activation affects multiple cell lineages present within the arthritic environment, therefore potentially acting as one of the drivers of disease pathogenesis. Since cell lineage-selective transgenic mouse models and specific Notch receptor inhibitors are becoming increasingly available, it can be expected that future research will evaluate whether Notch signaling components initiate crucial pathogenic impulses and, therefore, present viable therapeutic targets in inflammatory arthritis.

Spatial patterning during embryonic development emerges from the differentiation of progenitor cells that share the same genetic program. One of the main challenges in systems biology is to understand the relationship between gene network and patterning, especially how the cells communicate to coordinate their differentiation. This review aims to describe the principles of pattern formation from local cell-cell interactions mediated by the Notch signalling pathway. Notch mediates signalling via direct cell-cell contact and regulates cell fate decisions in many tissues during embryonic development. Here, I will describe the patterning mechanisms via different Notch ligands and the critical role of Notch oscillations during the segmentation of the vertebrate body, brain development, and blood vessel formation.

Notch signaling is a highly conserved pathway and it plays an essential role in regulating cellular proliferation, differentiation, and apoptosis. The human Notch family includes four receptors, Notch 1-4, and five ligands, delta-like ligand 1 (DLL1), delta-like ligand 3 (DLL3), delta-like ligand 4 (DLL4), Jagged-1 (JAG1), and Jagged-2 (JAG2). It is widely known, that Notch signaling components are often mutated and have deregulated expression in many types of cancer and other diseases. Thus, various therapeutic approaches targeting receptors and ligands of the Notch pathway are being investigated. Human JAG1 is closely related to tumor biology among the Notch ligands, and recent studies have shown potential for monoclonal antibodies targeting JAG1 in cancer therapy. Therefore, this review focuses on current reports on the significance of JAG1 directed cancer treatment, emphasizing immunotherapy.

Background/aim The DSL proteins, Serrate and Delta, which act as Notch receptor ligands, mediate signalling between adjacent cells, when a ligand-expressing cell binds to Notch on an adjacent receiving cell. Notch is ubiquitously expressed and DSL protein mis-expression can have devastating developmental consequences. Although transcriptional regulation of Delta and Serrate has been amply documented, we examined whether they are also regulated at the level of translation. Materials and methods We generated a series of deletions to investigate the initiation codon usage for Serrate using Drosophila S2 cells. Results Serrate mRNA contains three putative ATG initiation codons spanning a 60-codon region upstream of its signal peptide; we found that each one can act as an initiation codon, however, with a different translational efficiency. Conclusion Serrate expression is strictly regulated at the translational level.

Oncogenic signaling pathway reprograms cancer cell metabolism to promote aerobic glycolysis in favor of tumor growth. The ability of cancer cells to evade immunosurveillance and the role of metabolic regulators in T-cell functions suggest that oncogene-induced metabolic reprogramming may be linked to immune escape. Notch1 signaling, dysregulated in lung cancer, is correlated with increased glycolysis. Herein, we demonstrate in lung cancer that Notch1 promotes glycolytic gene expression through functional interaction with histone acetyltransferases p300 and pCAF. Notch1 signaling forms a positive feedback loop with TAZ. Notch1 transcriptional activity was increased in the presence of TAZ and the activation was TEAD1 independent. Notably, aerobic glycolysis was critical for Notch1/TAZ axis modulation of lung cancer growth in vitro and in vivo. Increased level of extracellular lactate via Notch1/TAZ axis inhibited cytotoxic T-cell activity, leading to the invasive characteristic of lung cancer cells. Interaction between Notch1 and TAZ promoted aerobic glycolysis and immune escape in lung cancer. Our findings provide potential therapeutic targets against Notch1 and TAZ and would be important for clinical translation in lung cancer.

Recent ability to derive endothelial cells (ECs) from induced pluripotent stem (iPS) cells holds a great therapeutic potential for personalized medicine and stem cell therapy. We aimed that better understanding of the complex molecular signals that are evoked during iPS cell differentiation toward ECs may allow specific targeting of their activities to enhance cell differentiation and promote tissue regeneration.In this study, we have generated mouse iPS cells from fibroblasts using established protocol. When iPS cells were cultivated on type IV mouse collagen-coated dishes in differentiation medium, cell differentiation toward vascular lineages were observed. To study the molecular mechanisms of iPS cell differentiation, we found that miR-199b is involved in EC differentiation. A step-wise increase in expression of miR-199 was detected during EC differentiation. Notably, miR-199b targeted the Notch ligand JAG1, resulting in vascular endothelial growth factor (VEGF) transcriptional activation and secretion through the transcription factor STAT3. Upon shRNA-mediated knockdown of the Notch ligand JAG1, the regulatory effect of miR-199b was ablated and there was robust induction of STAT3 and VEGF during EC differentiation. Knockdown of JAG1 also inhibited miR-199b-mediated inhibition of iPS cell differentiation toward smooth muscle markers. Using the in vitro tube formation assay and implanted Matrigel plugs, in vivo, miR-199b also regulated VEGF expression and angiogenesis.This study indicates a novel role for miR-199b as a regulator of the phenotypic switch during vascular cell differentiation derived from iPS cells by regulating critical signaling angiogenic responses. Stem Cells 2015;33:1405-1418.

Notch signaling regulates cell fate selection during development in multiple organs including the lung. Previous studies on the role of Notch in the lung focused mostly on Notch pathway core components or receptor-specific functions. It is unclear, however, how Jagged or Delta-like ligands collectively or individually (Jag1, Jag2, Dll1, Dll4) influence differentiation of airway epithelial progenitors. Using mouse genetic models we show major differences in Jag and Dll in regulation and establishment of cell fate. Jag ligands had a major impact in balancing distinct cell populations in conducting airways, but had no role in the establishment of domains and cellular abundance in the neuroendocrine (NE) microenvironment. Surprisingly, Dll ligands were crucial in restricting cell fate and size of NE bodies and showed an overlapping role with Jag in differentiation of NE-associated secretory (club) cells. These mechanisms may potentially play a role in human conditions that result in aberrant NE differentiation, including NE hyperplasias and cancer., eLife digest Cells communicate with each other by sending messages through a range of signaling pathways. One of the ways cells signal to each other is through a well-studied pathway known as Notch. In this pathway, cells display molecules on their surface, known as Notch ligands, that can activate Notch receptor proteins on the surface of neighboring cells. Once the Notch receptors bind to these ligands, they trigger various responses inside the cell. Notch ligands exist in two different families: Delta-like (Dll) ligands and Jagged (Jag) ligands. The layer of cells that lines the airways in the lungs consists of several different cell types. These include secretory cells that produce the fluid covering the airway surface, multiciliated cells, and neuroendocrine cells. Together these cells work as a barrier to protect the lung from environmental particles that may be breathed in. Additionally, the lung also has multipotent progenitor cells, which can become any of the other types. When Notch signaling is missing from the lung during embryonic development, not enough secretory cells are made, while other cell types are made in excess. This is because the multipotent progenitor cells need to communicate via Notch signaling to decide what type of cell to become and keep the right proportion of different cell types in the airways. In other organs, multipotent progenitors can become different types of cells depending on whether Notch signaling was activated by Dll or by Jag ligands, but it was unknown if this also happened in the lungs. Stupnikov et al. investigated the situation in the airways during development by looking at where and when Dll and Jag ligands first appeared, and by inactivating the genes that code for these ligands. They found that Jag ligands appeared well before Dll ligands, and that when the genes coding for Jag ligands were inactivated, more ciliated cells were produced. By contrast, loss of Dll ligands resulted in an increase in the neuroendocrine and their associated secretory cells, with little effect on the multiciliated cells. This increase resembled what is seen in some human diseases. The results suggest that the diversity of Notch effects in the airways depends on which Notch ligand is locally available. These observations may help to understand the mechanism of certain diseases involving neuroendocrine cells in the lung, such as small cell carcinoma or bronchial carcinoid tumors.

How alternative splicing (AS) is regulated in plants has not yet been elucidated. Previously, we have shown that the nuclear cap-binding protein complex (AtCBC) is involved in AS in Arabidopsis thaliana. Here we show that both subunits of AtCBC (AtCBP20 and AtCBP80) interact with SERRATE (AtSE), a protein involved in the microRNA biogenesis pathway. Moreover, using a high-resolution reverse transcriptase-polymerase chain reaction AS system we have found that AtSE influences AS in a similar way to the cap-binding complex (CBC), preferentially affecting selection of 5' splice site of first introns. The AtSE protein acts in cooperation with AtCBC: many changes observed in the mutant lacking the correct SERRATE activity were common to those observed in the cbp mutants. Interestingly, significant changes in AS of some genes were also observed in other mutants of plant microRNA biogenesis pathway, hyl1-2 and dcl1-7, but a majority of them did not correspond to the changes observed in the se-1 mutant. Thus, the role of SERRATE in AS regulation is distinct from that of HYL1 and DCL1, and is similar to the regulation of AS in which CBC is involved.

Size and shape constitute fundamental aspects in the description of morphology. Yet while the developmental-genetic underpinnings of trait size, in particular with regard to scaling relationships, are increasingly well understood, those of shape remain largely elusive. Here we investigate the potential function of the Notch signaling pathway in instructing the shape of beetle horns, a highly diversified and evolutionarily novel morphological structure. We focused on the bull-headed dung beetle Onthophagus taurus due to the wide range of horn sizes and shapes present among males in this species, in order to assess the potential function of Notch signaling in the specification of horn shape alongside the regulation of shape changes with allometry. Using RNA interference-mediated transcript depletion of Notch and its ligands, we document a highly conserved role of Notch signaling in general appendage formation. By integrating our functional genetic approach with a geometric morphometric analysis, we find that Notch signaling moderately but consistently affects horn shape, and does so differently for the horns of minor, intermediate-sized, and major males. Our results suggest that the function of Notch signaling during head horn formation may vary in a complex manner across male morphs, and highlights the power of integrating functional genetic and geometric morphometric approaches in analyzing subtle but nevertheless biologically important phenotypes in the face of significant allometric variation.

MicroRNAs (miRNA) are small RNAs of 20-22 nt that regulate diverse biological pathways through the modulation of gene expression. miRNAs recognize target RNAs by base complementarity and guide them to degradation or translational arrest. They are transcribed as longer precursors with extensive secondary structures. In plants, these precursors are processed by a complex harboring DICER-LIKE1 (DCL1), which cuts on the precursor stem region to release the mature miRNA together with the miRNA*. In both plants and animals, the miRNA precursors contain spatial clues that determine the position of the miRNA along their sequences. DCL1 is assisted by several proteins, such as the double-stranded RNA binding protein, HYPONASTIC LEAVES1 (HYL1), and the zinc finger protein SERRATE (SE). The precise biogenesis of miRNAs is of utter importance since it determines the exact nucleotide sequence of the mature small RNAs and therefore the identity of the target genes. miRNA processing itself can be regulated and therefore can determine the final small RNA levels and activity. Here, we describe methods to analyze miRNA processing intermediates in plants. These approaches can be used in wild-type or mutant plants, as well as in plants grown under different conditions, allowing a molecular characterization of the miRNA biogenesis from the RNA precursor perspective.

Purpose Delta-like ligand 4 (DLL4) and Jagged1 (JAG1), 2 vascular Notch ligands, are involved in the process of tumor angiogenesis. The present study investigates their relationship with microvascularization and the prognostic effect in primary glioblastoma. Methods Tumor tissues from 61 glioblastomas were analyzed using immunohistochemistry for DLL4/JAG1 expression and microvascular formations. The correlations between DLL4/JAG1 and microvascularization were analyzed. The survival probabilities were computed using the Kaplan-Meier method. The Cox proportional hazards regression model was used for multivariate analysis of time to progression (TTP) and overall survival (OS). Results The results showed increased DLL4 and JAG1 expression in glioblastoma tissues. Five types of basic microvascular formations, including microvascular sprouting, vascular cluster, vascular garland, glomeruloid vascular proliferation, and vasculogenic mimicry, were detected. Glioblastomas with the type I microvascular pattern (MVP) that displayed prominent microvascular sprouting and vascular clusters tended to have higher DLL4 expression, whereas those with the type II MVP that had numerous vascular garlands, glomeruloid vascular proliferations, and vasculogenic mimicries showed upregulated JAG1 expression. Univariate analysis documented that high DLL4 expression, high JAG1 expression, and type II (MVP) were statistically associated with reduced TTP and OS. Multivariate analysis confirmed high DLL4 expression, high JAG1 expression, and type II MVP as significant prognostic factors for both shorter TTP and OS, independent of age, Karnofsky performance scale, and other molecular markers (vascular endothelial growth factor, Ki67, and P53). Conclusions DLL4 and JAG1 may have opposing effects on tumor angiogenesis in glioblastoma. The Notch pathway may be a new target for antiangiogenic therapy in glioblastoma.

MicroRNAs (miRNAs) are small, noncoding ribonucleic acids that regulate gene expression by targeting mRNAs for translational repression and degradation. Accumulating experimental evidence supports a causal role of miRNAs in hematology tumorigenesis. However, the specific functions of miRNAs in the pathogenesis of multiple myeloma (MM) remain to be established. In this study, we demonstrated that miR-186 is commonly downregulated in MM cell lines and patient MM cells. Ectopic expression of miR-186 significantly inhibited cell growth, both in vitro and in vivo, and induced cell cycle G0/G1 arrest. Furthermore, miR-186 induced downregulation of Jagged1 protein expression by directly targeting its 3'-untranslated region (3'-UTR). Conversely, overexpression of Jagged1 rescued cells from miR-186-induced growth inhibition. Our collective results clearly indicate that miR-186 functions as a tumor suppressor in MM, supporting its potential as a therapeutic target for the disease.

Hematopoietic insufficiency is the hallmark of acute myeloid leukemia (AML) and predisposes patients to life-threatening complications such as bleeding and infections. Addressing the contribution of mesenchymal stromal cells (MSC) to AML-induced hematopoietic failure we show that MSC from AML patients (n=64) exhibit significant growth deficiency and impaired osteogenic differentiation capacity. This was molecularly reflected by a specific methylation signature affecting pathways involved in cell differentiation, proliferation and skeletal development. In addition, we found distinct alterations of hematopoiesis-regulating factors such as Kit-ligand and Jagged1 accompanied by a significantly diminished ability to support CD34+ hematopoietic stem and progenitor cells in long-term culture-initiating cells (LTC-ICs) assays. This deficient osteogenic differentiation and insufficient stromal support was reversible and correlated with disease status as indicated by Osteocalcin serum levels and LTC-IC frequencies returning to normal values at remission. In line with this, cultivation of healthy MSC in conditioned medium from four AML cell lines resulted in decreased proliferation and osteogenic differentiation. Taken together, AML-derived MSC are molecularly and functionally altered and contribute to hematopoietic insufficiency. Inverse correlation with disease status and adoption of an AML-like phenotype after exposure to leukemic conditions suggests an instructive role of leukemic cells on bone marrow microenvironment.

BACKGROUND Activation of Notch and NF-κB signaling has been frequently observed in colorectal cancer (CRC) patients and contributes to the chemo-resistance and treatment failure. However, the relationship between these signaling pathways and CRC has not been clearly described. OBJECTIVE To investigate the expression of Notch1, Jagged1, NF-κB and MMP-9 in CRC patients and analyze their correlation with clinicopathological factors. METHODS Expression of Notch1, Jagged1, NF-κB and MMP-9 was visualized by immune-histology in the tumor tissue, adjacent and distant normal tissues from 47 CRC patients without receiving chemotherapy or radiotherapy. RESULTS Notch1 (80.8%), Jagged1 (80.8%), NF-κB (70.2%) and MMP-9 (76.6%) were overexpressed in cancer tissues compared normal tissues (P< 0.05). The intensity of Notch1, Jagged1 and NF-κB expression was associated with histological grading, depth of invasion, TNM staging and lymph node metastasis of CRC. MMP-9 was intimately correlated with depth of invasion, TNM staging and lymph node metastasis. NF-κB, MMP-9 and Notch1 expression was positively correlated (P< 0.05), and the same positive correlation was found among NF-κB, MMP-9 and Jagged1 expression (P< 0.05). CONCLUSION Notch1, Jagged1, NF-κB and MMP-9 probably play a pivotal role during the CRC development, serving as biomarkers for early detection of the recurrence and metastasis and prognosis of CRC.

Prevailing dogma holds that cell-cell communication through Notch ligands and receptors determines binary cell fate decisions during progenitor cell divisions, with differentiated lineages remaining fixed. Mucociliary clearance in mammalian respiratory airways depends on secretory cells (club and goblet) and ciliated cells to produce and transport mucus. During development or repair, the closely related Jagged ligands (JAG1 and JAG2) induce Notch signalling to determine the fate of these lineages as they descend from a common proliferating progenitor. In contrast to such situations in which cell fate decisions are made in rapidly dividing populations, cells of the homeostatic adult airway epithelium are long-lived, and little is known about the role of active Notch signalling under such conditions. To disrupt Jagged signalling acutely in adult mammals, here we generate antibody antagonists that selectively target each Jagged paralogue, and determine a crystal structure that explains selectivity. We show that acute Jagged blockade induces a rapid and near-complete loss of club cells, with a concomitant gain in ciliated cells, under homeostatic conditions without increased cell death or division. Fate analyses demonstrate a direct conversion of club cells to ciliated cells without proliferation, meeting a conservative definition of direct transdifferentiation. Jagged inhibition also reversed goblet cell metaplasia in a preclinical asthma model, providing a therapeutic foundation. Our discovery that Jagged antagonism relieves a blockade of cell-to-cell conversion unveils unexpected plasticity, and establishes a model for Notch regulation of transdifferentiation.

Viroids are plant infecting, non - coding RNA molecules of economic importance. Potato spindle tuber viroid (PSTVd), the type species of Pospiviroidae family, has been shown to be affected by specific RNA silencing pathways. Dicer like 1 (DCL1), a key player in micro RNA (miRNA) pathway has been previously linked with PSTVd infectivity. In this report we aim to further dissect the interaction between the miRNA pathway and Pospiviroid virulence. We mainly focused on the Zinc-finger protein SERRATE (SE) a co-factor of DCL1 and core component of miRNA pathway. We generated Nicotiana tabacum and Nicotiana benthamiana SE knock-down plants exhibiting considerable miRNA reduction and strong phenotypic abnormalities. PSTVd infection of SE suppressed plants resulted in a significant viroid reduction, especially at the initial infection stages. This positive correlation between SE levels and viroid infectivity underlines its role in PSTVd life cycle and reveals the importance of the miRNA pathway upon viroid infection.

Yorki (Yki), a transcriptional co-activator that is a key component of the Hippo pathway, induces the transcription of a number of targets that promote cell proliferation and survival. Bombyx mori Yki3 (BmYki3), with 445 amino acid residues, facilitates cell migration and cell division, and enlarges cultured cell and wing disc size. In this study, cellular localization, transcriptional co-activator activity, cell migration, cell cycle, and cell size were characterized in alternative isoforms of BmYki. BmYki1 and BmYki3 are mainly located in the cytoplasm and nucleus, respectively, while, BmYki2 is located in both the cytoplasm and nucleus. The mutation BmYki1S97A (S97mutated to A) was transported from the cytoplasm to nucleus. Cell migration, cell cycle, and cell size could be enhanced by BmYki, however, the effect of BmYki1 and BmYki2 on cell proliferation was less compared to BmYki3. Moreover, wing discs could be enlarged by overexpressing BmYki1 or BmYki2 isoforms. Dual-luciferase reporter assay showed that BmYki3 had the highest activity to B. mori ovarian tumor gene. In BmN cells overexpressing one of the BmYki isoforms, expression levels of kibra ortholog (kibra), inhibitor of apoptosis protein (iap), four-jointed (fj), expanded (ex), crumbs (crb) and BMP and activin membrane-bound inhibitor homolog (Bmpr) genes were upregulated, while those of α-catenin (α-cat), decapentaplegic (dpp), serrate (serr) and signal transducer and activator of transcription (stat) genes were down-regulated. There was some difference in the regulation of gene expression between different isoforms. These results suggested that the activity of BmYki isoforms was different in the silkworm.

Basal cell carcinoma of the skin (BCC) is the most common skin tumor in Caucasians worldwide. Different therapeutic options are available to treat BCC, including topical immunotherapy. Imiquimod is topical Toll-like receptor 7 agonist that activates anti-tumor immune response and has been recently approved for the treatment of superficial BCC (sBCC). We sought to investigate the influence of imiquimod treatment on the members of the Notch signaling pathway, whose activity is known to be decreased in BCCs. Six patients with sBCC were evaluated for Notch1, Jagged1 and Delta1 expression before (pre-treatment) and after the beginning of the topical treatment (post-treatment) with imiquimod using real-time PCR and immunohistochemistry. We show selective transcriptional up-regulation of Notch pathway members (Notch1, Jagged1 and Delta1) in tumor cells of the sBCC post-treatment. Furthermore, we demonstrate minor increase of Notch1 protein expression on infiltrating cells as well as strong increase in Jagged1 protein expression in regressing sBCC tumors post-treatment. In this way, imiquimod may act as a stimulator of the Notch pathway in sBCC tumor cells by up-regulating protein expression of the Notch ligand, Jagged1. Via induction of Notch signaling imiquimod may exert tumor suppressor function, which together with its proinflammatory properties results in tumor regression.

Radiation exposure severely damages the hematopoietic system. Although several radio-protectors have been proposed to prevent radiation-induced damage, most agents have limited efficacy. In the present study, we investigated whether mesenchymal stem cells (MSCs) could contribute to the expansion of hematopoietic cells and mitigate radiation-induced hematopoietic injury in vitro and in vivo. We found that co-culture with MSCs promoted hematopoietic progenitor/stem cell (HPSCs) maintenance by providing a bone marrow-like microenvironment. In addition, we showed that MSCs prevented radiation-induced damage to HPSCs, as evidenced by the lack of DNA damage and apoptosis. Intravenously injected MSCs rapidly migrated to the bone marrow (BM) and prevented loss of BM cellularity, which reduced lethality and ameliorated pancytopenia in the BM of whole body-irradiated mice. We demonstrated that MSC-derived Jagged1 attenuated radiation-induced cytotoxicity of HPSCs, and that this was mediated by Notch signaling and expression of downstream proteins Bcl2 and p63 in HPSCs. In addition, Notch2 depletion significantly reduced the MSC-mediated radio-protective effect in human- and mouse-derived HPSCs. Collectively, our data show that activation of Notch and its associated downstream signaling pathways prevent radiation-induced hematopoietic injury. Therefore, enhancing Jagged1-Notch2 signaling could provide therapeutic benefit by protecting the hematopoietic system against damage after radiation.

The clinical use of doxorubicin (Dox) is limited by its cardiotoxicity. The fundamental changes it induces include interstitial myocardial fibrosis and the appearance of senescent cardiomyocytes. Mesenchymal stem cell (MSC)‑based therapies have also been reported to modulate cellular senescence, and have been used effectively to treat age‑related cardiovascular diseases. In the present study, the Transwell system was used to coculture H9c2 cells with MSCs, and their proliferation and viability were assessed. The expression of senescence‑related genes p53 and p16, and telomere length were measured using reverse transcription‑quantitative polymerase chain reaction analysis, and the Jagged‑1/Notch‑1 signaling pathway was detected using western blot analysis. The results revealed that Dox induced the senescence of H9c2 cells, characterized by a low proliferation rate, poor viability, reduced telomere length and impaired telomerase activity, and by marked increases in the expression of p53 and p16. By contrast, when cocultured with MSCs in the presence of Dox, H9c2 cell proliferation and viability increased, whereas the expression levels of p53 and p16 decreased, and telomere length and telomerase activity increased. The mechanism underlying the antisenescence function of MSCs was clarified, which involved the vascular endothelial growth factor (VEGF)/Jagged‑1/Notch‑1/transforming growth factor‑β1 (TGF‑β1) signaling pathway. It was confirmed that inhibiting VEGF, or silencing Jagged‑1 or Notch‑1 with small interfering RNA, or using recombinant TGF‑β1 eliminated the antisenescence effects of MSCs on the Dox‑treated H9c2 cells. The results revealed that MSCs rescued H9c2 cells from Dox‑induced senescence through the release of VEGF, which activated the Jagged‑1/Notch‑1 signaling pathway, leading to the inhibition of TGF‑β1 release. Therefore, treatment with MSCs may have important therapeutic implications on the attenuation of cardiotoxicity in patients with cancer treated with Dox.

T-cell development is a complex dynamic process that relies on ordered stromal signals delivered to thymus-seeding progenitors that migrate throughout different thymus microenvironments (TMEs). Particularly, Notch signaling provided by thymic epithelial cells (TECs) is crucial for T-cell fate specification and generation of mature T cells. Four canonical Notch ligands (Dll1, Dll4, Jag1 and Jag2) are expressed in the thymus, but their spatial distribution in functional TMEs is largely unknown, especially in humans, and their impact on Notch1 activation during T-lymphopoiesis remains undefined. Based on immunohistochemistry and quantitative confocal microscopy of fetal, postnatal and adult human and mouse thymus samples, we show that spatial regulation of Notch ligand expression defines discrete Notch signaling niches and dynamic species-specific TMEs. We further show that Notch ligand expression, particularly DLL4, is tightly regulated in cortical TECs during human thymus ontogeny and involution. Also, we provide the first evidence that NOTCH1 activation is induced in vivo in CD34 progenitors and developing thymocytes at particular cortical niches of the human fetal and postnatal thymus. Collectively, our results show that human thymopoiesis involves complex spatiotemporal regulation of Notch ligand expression, which ensures the coordinated delivery of niche-specific NOTCH1 signals required for dynamic T-cell development., Ministerio de Ciencia e Innovación [SAF2013-44857-R, SAF2014-62233-EXP and SAF2016-75442-R to M.L.T.]; Centros de Investigación Biomédica en Red [CB16/11/00399 to J.L.d.l.P.], Seventh Framework Programme [ThymiStem 602587 to M.L.T.] and Ministerio de Ciencia e Innovación [AP2007-01886 to M.J.G.-L.].

We have previously shown that an adverse perinatal environment significantly alters lung growth and development and results in persistently altered cardiopulmonary physiology in adulthood. Our model of maternal LPS treatment followed by 14 days of neonatal hyperoxia exposure causes severe pulmonary disease characterized by permanent decreases in alveolarization and diffuse interstitial fibrosis. The current investigations tested the hypothesis that dysregulation of Notch signaling pathways contributes to the permanently altered lung phenotype in our model and that the improvements we have observed previously with maternal docosahexaenoic acid (DHA) supplementation are mediated through normalization of Notch-related protein expression. Results indicated that inflammation (IL-6 levels) and oxidation (F2a-isoprostanes) persisted through 8 wk of life in mice exposed to LPS/O2 perinatally. These changes were attenuated by maternal DHA supplementation. Modest but inconsistent differences were observed in Notch-pathway proteins Jagged 1, DLL 1, PEN2, and presenilin-2. We detected substantial increases in markers of apoptosis including PARP-1, APAF-1, caspase-9, BCL2, and HMGB1, and these increases were attenuated in mice that were nursed by DHA-supplemented dams during the perinatal period. Although Notch signaling is not significantly altered at 8 wk of age in mice with perinatal exposure to LPS/O2, our findings indicate that persistent apoptosis continues to occur at 8 wk of age. We speculate that ongoing apoptosis may contribute to persistently altered lung development and may further enhance susceptibility to additional pulmonary disease. Finally, we found that maternal DHA supplementation prevented sustained inflammation, oxidation, and apoptosis in our model.

p53 and Notch-1 play important roles in breast cancer biology. Notch-1 inhibits p53 activity in cervical and breast cancer cells. Conversely, p53 inhibits Notch activity in T-cells but stimulates it in human keratinocytes. Notch co-activator MAML1 binds p53 and functions as a p53 co-activator. We studied the regulation of Notch signaling by p53 in MCF-7 cells and normal human mammary epithelial cells (HMEC). Results show that overexpression of p53 or activation of endogenous p53 with Nutlin-3 inhibits Notch-dependent transcriptional activity and Notch target expression in a dose-dependent manner. This effect could be partially rescued by transfection of MAML1 but not p300. Standard and quantitative co-immunoprecipitation experiments readily detected a complex containing p53 and Notch-1 in MCF-7 cells. Formation of this complex was inhibited by dominant negative MAML1 (DN-MAML1) and stimulated by wild-type MAML1. Standard and quantitative far-Western experiments showed a complex including p53, Notch-1, and MAML1. Chromatin immunoprecipitation (ChIP) experiments showed that p53 can associate with Notch-dependent HEY1 promoter and this association is inhibited by DN-MAML1 and stimulated by wild-type MAML1. Our data support a model in which p53 associates with the Notch transcriptional complex (NTC) in a MAML1-dependent fashion, most likely through a p53-MAML1 interaction. In our cellular models, the effect of this association is to inhibit Notch-dependent transcription. Our data suggest that p53-null breast cancers may lack this Notch-modulatory mechanism, and that therapeutic strategies that activate wild-type p53 can indirectly cause inhibition of Notch transcriptional activity.

Neurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder caused by impaired function of the neurofibromin RAS regulator. Using a combination of Nf1 genetically engineered mice and pharmacological/genetic inhibition approaches, we report that neurofibromin differentially controls neural stem cell (NSC) proliferation and multilineage differentiation through the selective use of the PI3K/AKT and RAF/MEK pathways. While PI3K/AKT governs neurofibromin-regulated NSC proliferation, multilineage differentiation is MEK-dependent. Moreover, whereas MEK-regulated multilineage differentiation requires Smad3-induced Jagged-1 expression and Notch activation, MEK/Smad3-regulated Hes1 induction is only responsible for astrocyte and neuronal differentiation. Collectively, these findings establish distinct roles for the RAS effector pathways in regulating brain NSC function.

The cytokine IL6 has a number of tumor-promoting activities in human and experimental cancers, but its potential as an angiogenic agent has not been fully investigated. Here, we show that IL6 can directly induce vessel sprouting in the ex vivo aortic ring model, as well as endothelial cell proliferation and migration, with similar potency to VEGF. However, IL6-stimulated aortic ring vessel sprouts had defective pericyte coverage compared with VEGF-stimulated vessels. The mechanism of IL6 action on pericytes involved stimulation of the Notch ligand Jagged1 as well as angiopoietin2 (Ang2). When peritoneal xenografts of ovarian cancer were treated with an anti-IL6 antibody, pericyte coverage of vessels was restored. In addition, in human ovarian cancer biopsies, there was an association between levels of IL6 mRNA, Jagged1, and Ang2. Our findings have implications for the use of cancer therapies that target VEGF or IL6 and for understanding abnormal angiogenesis in cancers, chronic inflammatory disease, and stroke. Cancer Res; 75(15); 3098–107. ©2015 AACR.

AIMS: Notch1 signalling in the heart is mainly activated via expression of Jagged1 on the surface of cardiomyocytes. Notch controls cardiomyocyte proliferation and differentiation in the developing heart and regulates cardiac remodelling in the stressed adult heart. Besides canonical Notch receptor activation in signal-receiving cells, Notch ligands can also activate Notch receptor-independent responses in signal-sending cells via release of their intracellular domain. We evaluated therefore the importance of Jagged1 (J1) intracellular domain (ICD)-mediated pathways in the postnatal heart. METHODS AND RESULTS: In cardiomyocytes, Jagged1 releases J1ICD, which then translocates into the nucleus and down-regulates Notch transcriptional activity. To study the importance of J1ICD in cardiac homeostasis, we generated transgenic mice expressing a tamoxifen-inducible form of J1ICD, specifically in cardiomyocytes. Using this model, we demonstrate that J1ICD-mediated Notch inhibition diminishes proliferation in the neonatal cardiomyocyte population and promotes maturation. In the neonatal heart, a response via Wnt and Akt pathway activation is elicited as an attempt to compensate for the deficit in cardiomyocyte number resulting from J1ICD activation. In the stressed adult heart, J1ICD activation results in a dramatic reduction of the number of Notch signalling cardiomyocytes, blunts the hypertrophic response, and reduces the number of apoptotic cardiomyocytes. Consistently, this occurs concomitantly with a significant down-regulation of the phosphorylation of the Akt effectors ribosomal S6 protein (S6) and eukaryotic initiation factor 4E binding protein1 (4EBP1) controlling protein synthesis. CONCLUSIONS: Altogether, these data demonstrate the importance of J1ICD in the modulation of physiological and pathological hypertrophy, and reveal the existence of a novel pathway regulating cardiac homeostasis.

Podocyte injury is an early feature of Fabry nephropathy, but the molecular mechanisms of podocyte injury are poorly understood. Lyso-Gb3 accumulates in serum in Fabry disease and increases extracellular matrix synthesis in podocytes. We explored the contribution of Notch1 signaling, a mediator of podocyte injury, to lyso-Gb3-elicited responses in cultured human podocytes. At clinically relevant concentrations, lyso-Gb3 activates podocyte Notch1 signaling, resulting in increased active Notch1 and HES1, a canonical Notch transcriptional target. A γ-secretase inhibitor or specific Notch1 small interfering RNA (siRNA) inhibited HES1 upregulation in response to lyso-Gb3. Notch1 siRNA or γ-secretase inhibition also prevented the lyso-Gb3-induced upregulation of Notch1, Notch ligand Jagged1 and chemokine (MCP1, RANTES) expression. Notch siRNA prevented the activation of nuclear factor kappa B (NFκB), and NFκB activation contributed to Notch1-mediated inflammatory responses as the NFκB inhibitor, parthenolide, prevented lyso-Gb3-induced chemokine upregulation. Notch1 also mediates fibrogenic responses in podocytes as Notch siRNA prevented lyso-Gb3 upregulation of fibronectin mRNA. Supporting the clinical relevance of cell culture findings, active Notch1, Jagged1 and HES1 were observed in Fabry kidney biopsies. Lyso-Gb3 elicited similar responses in mouse kidney. In conclusion, lyso-Gb3 promotes Notch1-mediated inflammatory and fibrogenic responses in podocytes that may contribute to Fabry nephropathy.

MicroRNAs are a class of small regulatory RNAs that are generated from primary miRNA (pri-miRNA) transcripts with a stem-loop structure. Accuracy of the processing of pri-miRNA into mature miRNA in plants can be enhanced by SERRATE (SE) and HYPONASTIC LEAVES 1 (HYL1). HYL1 activity is regulated by the FIERY2 (FRY2)/RNA polymerase II C-terminal domain phosphatase-like 1 (CPL1). Here, we discover that HIGH OSMOTIC STRESS GENE EXPRESSION 5 (HOS5) and two serine/arginine-rich splicing factors RS40 and RS41, previously shown to be involved in pre-mRNA splicing, affect the biogenesis of a subset of miRNA. These proteins are required for correct miRNA strand selection and the maintenance of miRNA levels. FRY2 dephosphorylates HOS5 whose phosphorylation status affects its subnuclear localization. HOS5 and the RS proteins bind both intronless and intron-containing pri-miRNAs. Importantly, all of these splicing-related factors directly interact with both HYL1 and SE in nuclear splicing speckles. Our results indicate that these splicing factors are directly involved in the biogenesis of a group of miRNA.

The subventricular zone is one of the 2 germinal niches of the adult brain where neural stem cells (NSC) generate new neurons and glia throughout life. NSC behavior is controlled by the integration of intrinsic signals and extrinsic cues provided by the surrounding microenvironment, or niche. Within the niche, the vasculature has emerged as a critical compartment, to which both neural stem cells and transit-amplifying progenitors are closely associated. A key function of the vasculature is to deliver blood-borne and secreted factors that promote proliferation and lineage progression of committed neural progenitors. We recently found that, in contrast to the established role of soluble cues, juxtacrine signals on vascular endothelial cells maintain neural stem cells in a quiescent and undifferentiated state through direct cell-cell interactions. In this perspective, we discuss how, through these apparently opposing signals, the vascular niche might coordinate stem cell decisions between maintenance and proliferation.

The aim of the present study was to examine the anticarcinogenic effects of emodin on the Notch signaling pathway and vascular endothelial growth factor (VEGF) in the PC3 androgen-independent prostate cancer cell line in vitro. The cell viability was assessed using an 3‑(4,5)‑dimethylthiahiazo(‑z‑y1)‑3,5‑di‑phenytetrazolium bromide assay. Cell apoptosis and cell cycle were detected using flow cytometry. Morphological alterations were observed using transmission electron microscopy. The mRNA and protein expression levels of Notch1, Jagged1, VEGF and bFGF were detected using reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. Laser scanning confocal microscope (LSCM) immunofluorescence analysis was performed to detect the levels of expression and the sub‑cellular localization of the Notch1 receptor protein. Growth of the PC3 cells was inhibited by emodin. Flow cytometry demonstrated that emodin induced apoptosis in the PC3 cells and arrested the cell cycle of the PC3 cells at the G2/M phase. The mRNA and protein expression of Notch1 in the PC3 cells was markedly increased, whereas the mRNA and protein expression levels of Jagged1, VEGF and bFGF were significantly decreased following exposure to emodin for 24 h. The LSCM assay revealed that the Notch1 was not only localized in the membrane and cytoplasm, but was also present in the nucleolus of the PC3 cells, and the expression of Notch1 in the nuclei gradually increased following treatment with emodin. These results demonstrated that emodin suppressed the growth of androgen‑independent prostate cancer cell lines and induced apoptosis and cell cycle arrest. The Notch signaling pathway was activated in the PC3 cells following exposure to emodin, which suggested that the Notch signaling pathway is involved in the prostate tumor‑suppressing mechanisms of emodin, the activation of which may depend on CBF1 protein in the nucleus by classic pathways. The antitumor function of emodin, attributed to the Notch signaling pathway, induced the downregulation of VEGF by suppressing tumorigenesis and angiogenesis, which indicated a novel mechanism underlying the emodin-mediated anti-prostate cancer effect.

// Ana-Rita Pedrosa 1 , Alexandre Trindade 1,2 , Catarina Carvalho 1 , Jose Graca 1 , Sandra Carvalho 1 , Maria C. Peleteiro 1 , Ralf H. Adams 3,4 and Antonio Duarte 1,2 1 Centro Interdisciplinar de Investigacao em Sanidade Animal (CIISA), University of Lisbon, Lisbon, Portugal 2 Instituto Gulbenkian de Ciencia, Oeiras, Portugal 3 Department of Tissue Morphogenesis, Max Planck Institute for Molecular Biomedicine, Muenster, Germany 4 Faculty of Medicine, University of Muenster, Muenster, Germany Correspondence to: Antonio Duarte, email: // Alexandre Trindade, email: // Keywords : Jagged1, Notch, TRAMP, tumor angiogenesis, angiocrine Received : May 21, 2015 Accepted : May 29, 2015 Published : June 08, 2015 Abstract Angiogenesis is an essential process required for tumor growth and progression. The Notch signaling pathway has been identified as a key regulator of the neo-angiogenic process. Jagged-1 ( Jag1 ) is a Notch ligand required for embryonic and retinal vascular development, which direct contribution to the regulation of tumor angiogenesis remains to be fully characterized. The current study addresses the role of endothelial Jagged1-mediated Notch signaling in the context of tumoral angiogenesis in two different mouse tumor models: subcutaneous Lewis Lung Carcinoma (LLC) tumor transplants and the autochthonous Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP). The role of endothelial Jagged1 in tumor growth and neo-angiogenesis was investigated with endothelial-specific Jag1 gain- and loss-of-function mouse mutants ( eJag1 OE and eJag1c KO). By modulating levels of endothelial Jag1 , we observed that this ligand regulates tumor vessel density, branching, and perivascular maturation, thus affecting tumor vascular perfusion. The pro-angiogenic function is exerted by its ability to positively regulate levels of Vegfr-2 while negatively regulating Vegfr-1 . Additionally, endothelial Jagged1 appears to exert an angiocrine function possibly by activating Notch3/Hey1 in tumor cells, promoting proliferation, survival and epithelial-to-mesenchymal transition (EMT), potentiating tumor development. These findings provide valuable mechanistic insights into the role of endothelial Jagged1 in promoting solid tumor development and support the notion that it may constitute a promising target for cancer therapy.

Proliferative vitreoretinopathy (PVR) is the major cause of failure in patients undergoing surgery for rhegmatogenous retinal detachment (RRD). Characterized by the formation of an abnormal contractile membrane within the eye, PVR can cause tractional retinal redetachment. Epithelial-to-mesenchymal transition (EMT), in which epithelial cells morphologically and phenotypically transdifferentiate into mesenchymal cells, is the major pathological process implicated in PVR. Among the various cell types involved in the process, retinal pigment epithelium cells are primary contributors although, after decades of research, the mechanisms underlying EMT have remained elusive. Recently, signaling pathways, some involving growth factors, have been demonstrated to contribute to EMT. In this article, we review research to date about the roles of such signaling, including including transforming growth factor-beta-, hepatocyte growth factor-, platelet-derived growth factor-, and Notch-, Wnt/β-catenin-, and Hippo-signaling pathways, in the EMT of PVR.

Acute myelogenous leukemia (AML) is a heterogeneous disorder characterized by clonal proliferation of stem cell-like blasts in bone marrow (BM); however, their unique cellular interaction within the BM microenvironment and its functional significance remain unclear. Here, we assessed the BM microenvironment of AML patients and demonstrate that the leukemia stem cells induce a change in the transcriptional programming of the normal mesenchymal stromal cells (MSC). The modified leukemic niche alters the expressions of cross-talk molecules (i.e., CXCL12 and JAG1) in MSCs to provide a distinct cross-talk between normal and leukemia cells, selectively suppressing normal primitive hematopoietic cells while supporting leukemogenesis and chemoresistance. Of note, AML patients exhibited distinct heterogeneity in the alteration of mesenchymal stroma in BM. The distinct pattern of stromal changes in leukemic BM at initial diagnosis was associated with a heterogeneous posttreatment clinical course with respect to the maintenance of complete remission for 5 to 8 years and early or late relapse. Thus, remodeling of mesenchymal niche by leukemia cells is an intrinsic self-reinforcing process of leukemogenesis that can be a parameter for the heterogeneity in the clinical course of leukemia and hence serve as a potential prognostic factor. Cancer Res; 75(11); 2222–31. ©2015 AACR.

Aims Notch signalling is essential for blood vessel formation. During angiogenesis, the Notch ligand DLL4 on the leading tip cell activates Notch receptors on the adjacent stalk cells. DLL4-Notch signalling is impaired by the Notch ligand JAG1 in endothelial cells. The Delta/Serrate/Lag2 (DSL) domain of the Notch ligands binds to the EGF-like repeats 11–13 of the Notch receptor. This study aimed to elucidate how soluble proteins containing these short domains interfere with Notch signalling during angiogenesis. Methods and results Adenoviral vectors were generated to express the DSL domains of DLL1, DLL4, JAG1, and the Notch1 EGF-like repeats 11–13 fused to immunoglobulin-G heavy chain. These soluble ligand peptides inhibited Notch signalling in endothelial cells and this caused hyperbranching in cellular angiogenesis assays and in the neonatal mouse retina. The soluble Notch receptor peptides bound stronger to JAG1 than DLL4 ligands, resulting in increased signalling activity. This led to impaired tip cell formation and less vessel sprouting in the retina. Conclusion The minimal binding domains of Notch ligands are sufficient to interfere with Notch signalling. The corresponding soluble Notch1 EGF11-13 peptide binds stronger to inhibitory Notch ligands and thereby promotes Notch signalling in endothelial cells.

The Notch pathway is a fundamental signalling system in most multicellular animals. We have determined the X-ray crystal structure of the extracellular domain of the Notch ligand delta-like ligand-1 (Dll-1). The structure incorporates the N-terminal C2 domain, receptor-binding DSL domain and the first six (of eight) EGF (epidermal growth factor)-like repeats, which form a highly extended conformation, confirmed by analytical ultracentrifugation. Comparison of our structure with a fragment of Jagged1 ligand allows us to dissect the similarities and differences between the ligand families. Differences in the C2 domains of Dll-1 and Jagged1 suggest their lipid-binding properties are likely to differ. A conserved hydrophobic patch on the surface of both Dll-1 and Jagged1 provides a likely receptor-interaction site that is common to both ligands. We also explore the binding affinity of Dll-1 for a fragment of Notch1 using different techniques. Apparent binding affinities vary when different techniques are used, explaining discrepancies in the literature. Using analytical ultracentrifugation, we perform for the first time binding analyses where both receptor and ligand are in solution, which confirms a Kd of 10 μM for this interaction.

Objective— Notch signaling controls cardiovascular development and has been associated with several pathological conditions. Among its ligands, Jagged1 and Dll4 were shown to have opposing effects in developmental angiogenesis, but the underlying mechanism and the role of Jagged1/Notch signaling in adult angiogenesis remain incompletely understood. The current study addresses the importance of endothelial Jagged1-mediated Notch signaling in the context of adult physiological angiogenesis and the interactions of Jagged1 and Dll4 on angiogenic response and vascular maturation processes. Approach and Results— The role of endothelial Jagged1 in wound healing kinetics and angiogenesis was investigated with endothelial-specific Jag1 gain-of-function and loss-of-function mouse mutants ( eJag1 OE and eJag1c KO). To study the interactions between the 2 Notch ligands, genetic mouse models were combined with pharmacological inhibition of Dll4 or Jagged1, respectively. Jagged1 overexpression in endothelial cells increased vessel density, maturation, and perfusion, thus accelerating wound healing. The opposite effect was seen in e Jag1 cKO animals. Interestingly, Dll4 blockade in these animals led to an increase in vascular density but induced a greater decrease in perivascular cell coverage. However, Jagged1 inhibition in Dll4 gain-of-function (e Dll4 OE) mutants, with reduced angiogenesis, further diminished angiogenic growth and hampered perivascular cell coverage. Our findings suggest that as Dll4 blocks endothelial activation through Notch1 signaling, it also induces Jagged1 expression. Jagged1 then blocks Dll4 signaling through Notch1, allowing endothelial activation by vascular endothelial growth factor and endothelial layer growth. Jagged1 also initiates maturation of the newly formed vessels, possibly by binding and activating endothelial Notch4. Importantly, mice administered with a Notch4 agonistic antibody mimicked the mural cell phenotype of e Jag1 OE mutants without affecting angiogenic growth, which is thought to be Notch1 dependent. Conclusions— Endothelial Jagged1 is likely to operate downstream of Dll4/Notch1 signaling to activate Notch4 and regulate vascular maturation. Thus, Jagged1 not only counteracts Dll4/Notch in the endothelium but also generates a balance between angiogenic growth and maturation processes in vivo.

TNF-α inhibitor reportedly protects against myocardial ischemia/reperfusion (MI/R) injury. It can also increase Notch1 expression in inflammatory bowel disease, revealing the regulation of Notch1 signaling by TNF-α inhibitor. However, the interaction between TNF-α inhibitor and Notch1 signaling in MI/R remains unclear. This study aimed to determine the involvement of TNF-α inhibitor with Notch1 in MI/R and delineate the related mechanism. Notch1-specific small interfering RNA (20 μg) or Jagged1 (a Notch ligand, 12 μg) was delivered through intramyocardial injection. Forty-eight hours after injection, mice received 30 min of myocardial ischemia followed by 3 h (for cell apoptosis and oxidative/nitrative stress) or 24h (for infarct size and cardiac function) of reperfusion. Ten minutes before reperfusion, mice randomly received an intraperitoneal injection of vehicle, etanercept, diphenyleneiodonium, 1400W, or EUK134. Finally, downregulation of Notch1 significantly reversed the alleviation of MI/R injury induced by etanercept, as evidenced by enlarged myocardial infarct size, suppressed cardiac function, and increased myocardial apoptosis. Moreover, Notch1 blockade increased the expression of inducible NO synthase (iNOS) and gp(91)(phox), enhanced NO and superoxide production, and accelerated their cytotoxic reaction product, peroxynitrite. Furthermore, NADPH inhibition with diphenyleneiodonium or iNOS suppression with 1400W mitigated the aggravation of MI/R injury induced by Notch1 downregulation in mice treated with etanercept. Additionally, either Notch1 activation with Jagged1 or peroxynitrite decomposition with EUK134 reduced nitrotyrosine content and attenuated MI/R injury. These data indicate that MI/R injury can be attenuated by TNF-α inhibitor, partly via Notch1 signaling-mediated suppression of oxidative/nitrative stress.

Notch signaling has been shown to contribute to murine pancreatic development at various stages. Delta-like 1 (Dll1) or Jagged1 (Jag1) are the Notch ligands that solely function to trigger this signaling during the pancreatic bud stage (~e9.5) or after birth, respectively. However, it has not been elucidated whether these Notch ligands are required at the later stage (e10.5-18.5) when the particular pancreas structures form. Here, we detected the dual expression of Dll1 and Jag1 in the epithelium after e10.5, which was restricted to the ductal cell lineage, including centroacinar cells expressing Sox9, CD133 and Hes1 but not the ductal cell markers Hnf1β and DBA, at e18.5. To evaluate the significance of the Notch ligands during this period, we established double-floxed mice of Dll1 and Jag1 genes with Ptf1a-Cre knock-in allele and examined the effects on development. The abrogation of both ligands but not a single one led to the loss of centroacinar cells, which was due to the decrease in cell proliferation and the increase in cell death, as well as to the reduction of Sox9. These results suggested that Dll1 and Jag1 function redundantly and are necessary to maintain the centroacinar cells as an environmental niche in the developing pancreas.

Hematopoietic stem cells (HSCs) reside in regulatory niches in the bone marrow (BM). Although HSC niches have been extensively characterized, the role of endosteal osteoblasts (OBs) in HSC regulation requires further clarification, and the role of OBs in regulating leukemic stem cells (LSCs) is not well studied. We used an OB visualization and ablation mouse model to study the role of OBs in regulating normal HSCs and chronic myelogenous leukemia (CML) LSCs. OB ablation resulted in increase in cells with a LSK Flt3(-)CD150(+)CD48(-) long-term HSC (LTHSC) phenotype but reduction of a more highly selected LSK Flt3(-)CD34(-)CD49b(-)CD229(-) LTHSC subpopulation. LTHSCs from OB-ablated mice demonstrated loss of quiescence and reduced long-term engraftment and self-renewal capacity. Ablation of OB in a transgenic CML mouse model resulted in accelerated leukemia development with reduced survival compared with control mice. The notch ligand Jagged-1 was overexpressed on CML OBs. Normal and CML LTHSCs cultured with Jagged-1 demonstrated reduced cell cycling, consistent with a possible role for loss of Jagged-1 signals in altered HSC and LSC function after OB ablation. These studies support an important role for OBs in regulating quiescence and self-renewal of LTHSCs and a previously unrecognized role in modulating leukemia development in CML.